Methods for Adding Adapters to Nucleic Acids and Compositions for Practicing the Same

ABSTRACT

Provided are methods of adding adapters to nucleic acids. The methods include combining in a reaction mixture a template ribonucleic acid (RNA), a template switch oligonucleotide including a 3′ hybridization domain and a sequencing platform adapter construct, a polymerase, and dNTPs. The reaction mixture components are combined under conditions sufficient to produce a product nucleic acid that includes the template RNA and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid that includes a region polymerized from the dNTPs by the polymerase. Aspects of the invention further include compositions and kits.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/469,364 filed Mar. 24, 2017; which application is a continuation ofU.S. application Ser. No. 14/478,978 filed Sep. 5, 2014; whichapplication, pursuant to 35 U.S.C. § 119 (e), claims priority to thefiling date of the U.S. Provisional Patent Application Ser. No.61/892,372 filed Oct. 17, 2013 and U.S. Provisional Patent ApplicationSer. No. 61/979,852 filed Apr. 15, 2014; the disclosures of whichapplications are herein incorporated by reference.

INTRODUCTION

Massively parallel (or “next generation”) sequencing platforms arerapidly transforming data collection and analysis in genome, epigenomeand transcriptome research. Certain sequencing platforms, such as thosemarketed by Illumina®, Ion Torrent™, Roche™, and Life Technologies™,involve solid phase amplification of polynucleotides of unknownsequence. Solid phase amplification of these polynucleotides istypically performed by first ligating known adapter sequences to eachend of the polynucleotide. The double-stranded polynucleotide is thendenatured to form a single-stranded template molecule. The adaptersequence on the 3′ end of the template is hybridized to an extensionprimer that is immobilized on the solid substrate, and amplification isperformed by extending the immobilized primer. In what is often referredto as “bridge PCR”, a second immobilized primer, identical to the 5′ endof the template, serves as a reverse primer, allowing amplification ofboth the forward and reverse strands to proceed on the solid substrate,e.g., a bead or surface of a flow cell.

A disadvantage of ligation-based approaches for sequencing adapteraddition is the number of steps involved, including the enzymatic andwash steps that are needed to prepare the target polynucleotide beforesolid phase amplification can be initiated. As one example, afterligation of the adapter sequences, unused adapter molecules must beseparated from the ligated polynucleotides before adding the mixture tothe flow cell. Otherwise, the unused adapter molecules can alsohybridize to the immobilized primers, preventing efficient hybridizationof the primers to the template molecules and subsequent extension.

An additional drawback of ligation-based approaches is their lack ofdirectionality, which makes it difficult to have different adapters atthe different ends of the nucleic acids. Moreover, the sensitivity ofsuch methods is low and renders them unsuitable under circumstanceswhere only a small amount of sample material is available.

SUMMARY

Provided are methods of adding adapters to nucleic acids. The methodsinclude combining in a reaction mixture a template ribonucleic acid(RNA), a template switch nucleic acid (e.g., a template switcholigonucleotide) including a 3′ hybridization domain and a sequencingplatform adapter construct, a polymerase, and dNTPs. The reactionmixture components are combined under conditions sufficient to produce aproduct nucleic acid that includes the template RNA and the templateswitch oligonucleotide each hybridized to adjacent regions of a singleproduct nucleic acid that includes a region polymerized from the dNTPsby the polymerase. Aspects of the invention further include compositionsand kits.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates a template switch-based method forgenerating a nucleic acid having adapter constructs according to oneembodiment of the present disclosure. In this embodiment, adapterconstructs having less than all nucleic acid domains necessary for asequencing platform of interest are provided by a template-switchpolymerization reaction. The remaining nucleic acid domains are providedby polymerase chain reaction (PCR) using amplification primers thatinclude the remaining domains.

FIG. 2 schematically illustrates a template switch-based method forgenerating a nucleic acid having adapter constructs according to oneembodiment of the present disclosure. In this embodiment, adapters thatinclude all nucleic acid domains necessary for a sequencing platform ofinterest are provided during a template-switch polymerization reaction.

FIG. 3 schematically illustrates a template switch-based method forgenerating a nucleic acid having adapter constructs according to oneembodiment of the present disclosure. In this embodiment,non-polyadenylated RNA is used as the starting material. Thenon-polyadenylated RNA is adenylated, and the adenylated RNA serves asthe donor template in a template-switch polymerization reaction thatgenerates a nucleic acid having adapter constructs. From top to bottom,SEQ ID NOs:11-12.

FIG. 4 is a graph showing that a cDNA library may be generated using themethods of the present disclosure with various amounts of input RNA.According to this embodiment, the cDNAs that make up the library haveadapter constructs that enable sequencing of the cDNAs by a sequencingplatform of interest.

FIG. 5 shows adapter constructs according to one embodiment of thepresent disclosure. In this embodiment, the constructs include the P5,P7, Read 1, Read 2, and index domains which enable paired-end sequencingof a cDNA corresponding to a template RNA on an Illumina® sequencingplatform. From top to bottom, SEQ ID NOs:8-10.

FIG. 6 shows a comparison of sequencing data generated using a methodaccording to one embodiment of the present disclosure and sequencingdata generated using the traditional method of separate cDNAamplification and library preparation steps.

DETAILED DESCRIPTION

Provided are methods of adding adapters to nucleic acids. The methodsinclude combining in a reaction mixture a template ribonucleic acid(RNA), a template switch oligonucleotide including a 3′ hybridizationdomain and a sequencing platform adapter construct, a polymerase, anddNTPs. The reaction mixture components are combined under conditionssufficient to produce a product nucleic acid that includes the templateRNA and the template switch oligonucleotide each hybridized to adjacentregions of a single product nucleic acid that includes a regionpolymerized from the dNTPs by the polymerase. Aspects of the inventionfurther include compositions and kits.

Before the methods of the present disclosure are described in greaterdetail, it is to be understood that the methods are not limited toparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the methods will be limited only bythe appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the methods. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the methods, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the methods.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods belong. Although any methods similar orequivalent to those described herein can also be used in the practice ortesting of the methods, representative illustrative methods andmaterials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present methods are not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the methods, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the methods, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace operableprocesses and/or devices/systems/kits. In addition, all sub-combinationslisted in the embodiments describing such variables are alsospecifically embraced by the present methods and are disclosed hereinjust as if each and every such sub-combination was individually andexplicitly disclosed herein.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentmethods. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Methods

Methods of adding adapters to nucleic acids are provided. The methodsutilize the ability of certain nucleic acid polymerases to “templateswitch,” using a first ribonucleic acid (RNA) strand as a template forpolymerization, and then switching to a second template nucleic acidstrand (which may be referred to as a “template switch nucleic acid” oran “acceptor template”) while continuing the polymerization reaction.The result is the synthesis of a hybrid nucleic acid strand with a 5′region complementary to the first template nucleic acid strand and a 3′region complementary to the template switch nucleic acid. In certainaspects, the nucleotide sequence of all or a portion (e.g., a 5′ region)of the template switch oligonucleotide may be defined by a practitionerof the subject methods such that the newly-synthesized hybrid nucleicacid strand has a partial or complete sequencing platform adaptersequence at its 3′ end useful for sequencing the hybrid nucleic acidstrand using a sequencing platform of interest. Sequencing platforms ofinterest include, but are not limited to, the HiSeq™, MiSeq™ and GenomeAnalyzer™ sequencing systems from Illumina®; the Ion PGM™ and IonProton™ sequencing systems from Ion Torrent™; the PACBIO RS IIsequencing system from Pacific Biosciences, the SOLiD sequencing systemsfrom Life Technologies™, the 454 GS FLX+ and GS Junior sequencingsystems from Roche, or any other sequencing platform of interest.

In certain aspects, the polymerization reaction is initiated using aprimer having a partial or complete sequencing platform adapter sequenceat its 5′ end, resulting in a hybrid nucleic acid strand having apartial or complete sequencing platform adapter sequence at each end.The directionality of the adapters in the hybrid nucleic acid strand maybe predetermined by a practitioner of the subject methods, e.g., byselecting the adapter sequence present at the 5′ end of the primer, andthe adapter sequence present at the 5′ end of the template switcholigonucleotide. Here, the adapter sequence present in the primer andthe adapter sequence in the template switch oligonucleotide will bepresent at the 5′ and 3′ ends of the hybrid nucleic acid strand,respectively.

According to the methods of the present disclosure, the reaction mixturecomponents are combined under conditions sufficient to produce a productnucleic acid that includes the template RNA and the template switcholigonucleotide each hybridized to adjacent regions of a single productnucleic acid that includes a region polymerized from the dNTPs by thepolymerase.

By “conditions sufficient to produce a product nucleic acid” is meantreaction conditions that permit polymerase-mediated extension of a 3′end of a nucleic acid strand hybridized to the template RNA, templateswitching of the polymerase to the template switch oligonucleotide, andcontinuation of the extension reaction using the template switcholigonucleotide as the template. Achieving suitable reaction conditionsmay include selecting reaction mixture components, concentrationsthereof, and a reaction temperature to create an environment in whichthe polymerase is active and the relevant nucleic acids in the reactioninteract (e.g., hybridize) with one another in the desired manner. Forexample, in addition to the template RNA, the polymerase, the templateswitch oligonucleotide and dNTPs, the reaction mixture may includebuffer components that establish an appropriate pH, salt concentration(e.g., KCl concentration), metal cofactor concentration (e.g., Mg²⁺ orMn²⁺ concentration), and the like, for the extension reaction andtemplate switching to occur. Other components may be included, such asone or more nuclease inhibitors (e.g., an RNase inhibitor and/or a DNaseinhibitor), one or more additives for facilitatingamplification/replication of GC rich sequences (e.g., GC-Melt™ reagent(Clontech Laboratories, Inc. (Mountain View, Calif.)), betaine, DMSO,ethylene glycol, 1,2-propanediol, or combinations thereof), one or moremolecular crowding agents (e.g., polyethylene glycol, or the like), oneor more enzyme-stabilizing components (e.g., DTT present at a finalconcentration ranging from 1 to 10 mM (e.g., 5 mM)), and/or any otherreaction mixture components useful for facilitating polymerase-mediatedextension reactions and template-switching.

The reaction mixture can have a pH suitable for the primer extensionreaction and template-switching. In certain embodiments, the pH of thereaction mixture ranges from 5 to 9, such as from 7 to 9, including from8 to 9, e.g., 8 to 8.5. In some instances, the reaction mixture includesa pH adjusting agent. pH adjusting agents of interest include, but arenot limited to, sodium hydroxide, hydrochloric acid, phosphoric acidbuffer solution, citric acid buffer solution, and the like. For example,the pH of the reaction mixture can be adjusted to the desired range byadding an appropriate amount of the pH adjusting agent.

The temperature range suitable for production of the product nucleicacid may vary according to factors such as the particular polymeraseemployed, the melting temperatures of any optional primers employed,etc. According to one embodiment, the polymerase is a reversetranscriptase (e.g., an MMLV reverse transcriptase) and the reactionmixture conditions sufficient to produce the product nucleic acidinclude bringing the reaction mixture to a temperature ranging from 4°C. to 72° C., such as from 16° C. to 70° C., e.g., 37° C. to 50° C.,such as 40° C. to 45° C., including 42° C.

The template ribonucleic acid (RNA) may be a polymer of any lengthcomposed of ribonucleotides, e.g., 10 bases or longer, 20 bases orlonger, 50 bases or longer, 100 bases or longer, 500 bases or longer,1000 bases or longer, 2000 bases or longer, 3000 bases or longer, 4000bases or longer, 5000 bases or longer or more bases. In certain aspects,the template ribonucleic acid (RNA) is a polymer composed ofribonucleotides, e.g., 10 bases or less, 20 bases or less, 50 bases orless, 100 bases or less, 500 bases or less, 1000 bases or less, 2000bases or less, 3000 bases or less, 4000 bases or less, or 5000 bases orless. The template RNA may be any type of RNA (or sub-type thereof)including, but not limited to, a messenger RNA (mRNA), a microRNA(miRNA), a small interfering RNA (siRNA), a transacting smallinterfering RNA (ta-siRNA), a natural small interfering RNA (nat-siRNA),a ribosomal RNA (rRNA), a transfer RNA (tRNA), a small nucleolar RNA(snoRNA), a small nuclear RNA (snRNA), a long non-coding RNA (lncRNA), anon-coding RNA (ncRNA), a transfer-messenger RNA (tmRNA), a precursormessenger RNA (pre-mRNA), a small Cajal body-specific RNA (scaRNA), apiwi-interacting RNA (pi RNA), an endoribonuclease-prepared si RNA(esiRNA), a small temporal RNA (stRNA), a signal recognition RNA, atelomere RNA, a ribozyme, or any combination of RNA types thereof orsubtypes thereof.

The RNA sample that includes the template RNA may be combined into thereaction mixture in an amount sufficient for producing the productnucleic acid. According to one embodiment, the RNA sample is combinedinto the reaction mixture such that the final concentration of RNA inthe reaction mixture is from 1 fg/μL to 10 μg/μL, such as from 1 pg/μLto 5 μg/μL, such as from 0.001 μg/μL to 2.5 μg/μL, such as from 0.005μg/μL to 1 μg/μL, such as from 0.01 μg/μL to 0.5 μg/μL, including from0.1 μg/μL to 0.25 μg/μL. In certain aspects, the RNA sample thatincludes the template RNA is isolated from a single cell. In otheraspects, the RNA sample that includes the template RNA is isolated from2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 20 or more, 50 or more, 100 or more,or 500 or more cells. According to certain embodiments, the RNA samplethat includes the template RNA is isolated from 500 or less, 100 orless, 50 or less, 20 or less, 10 or less, 9, 8, 7, 6, 5, 4, 3, or 2cells.

The template RNA may be present in any nucleic acid sample of interest,including but not limited to, a nucleic acid sample isolated from asingle cell, a plurality of cells (e.g., cultured cells), a tissue, anorgan, or an organism (e.g., bacteria, yeast, or the like). In certainaspects, the nucleic acid sample is isolated from a cell(s), tissue,organ, and/or the like of a mammal (e.g., a human, a rodent (e.g., amouse), or any other mammal of interest). In other aspects, the nucleicacid sample is isolated from a source other than a mammal, such asbacteria, yeast, insects (e.g., drosophila), amphibians (e.g., frogs(e.g., Xenopus)), viruses, plants, or any other non-mammalian nucleicacid sample source.

Approaches, reagents and kits for isolating RNA from such sources areknown in the art. For example, kits for isolating RNA from a source ofinterest—such as the NucleoSpin®, NucleoMag® and NucleoBond® RNAisolation kits by Clontech Laboratories, Inc. (Mountain View,Calif.)—are commercially available. In certain aspects, the RNA isisolated from a fixed biological sample, e.g., formalin-fixed,paraffin-embedded (FFPE) tissue. RNA from FFPE tissue may be isolatedusing commercially available kits—such as the NucleoSpin® FFPE RNA kitsby Clontech Laboratories, Inc. (Mountain View, Calif.).

In certain aspects, the subject methods include producing the templateRNA from a precursor RNA. For example, when it is desirable to controlthe size of the template RNA that is combined into the reaction mixture,an RNA sample isolated from a source of interest may be subjected toshearing/fragmentation, e.g., to generate a template RNA that is shorterin length as compared to a precursor non-sheared RNA (e.g., afull-length mRNA) in the original sample. The template RNA may begenerated by a shearing/fragmentation strategy including, but notlimited to, passing the sample one or more times through a micropipettetip or fine-gauge needle, nebulizing the sample, sonicating the sample(e.g., using a focused-ultrasonicator by Covaris, Inc. (Woburn, Mass.)),bead-mediated shearing, enzymatic shearing (e.g., using one or moreRNA-shearing enzymes), chemical based fragmentation, e.g., usingdivalent cations, fragmentation buffer (which may be used in combinationwith heat) or any other suitable approach for shearing/fragmenting aprecursor RNA to generate a shorter template RNA. In certain aspects,the template RNA generated by shearing/fragmentation of a startingnucleic acid sample has a length of from 10 to 20 nucleotides, from 20to 30 nucleotides, from 30 to 40 nucleotides, from 40 to 50 nucleotides,from 50 to 60 nucleotides, from 60 to 70 nucleotides, from 70 to 80nucleotides, from 80 to 90 nucleotides, from 90 to 100 nucleotides, from100 to 150 nucleotides, from 150 to 200, from 200 to 250 nucleotides inlength, or from 200 to 1000 nucleotides or even from 1000 to 10,000nucleotides, for example, as appropriate for the sequencing platformchosen.

Additional strategies for producing the template RNA from a precursorRNA may be employed. For example, producing the template RNA may includeadding nucleotides to an end of the precursor RNA. In certain aspects,the precursor RNA is a non-polyadenylated RNA (e.g., a microRNA, smallRNA, or the like), and producing the template RNA includes adenylating(e.g., polyadenylating) the precursor RNA. Adenylating the precursor RNAmay be performed using any convenient approach. According to certainembodiments, the adenylation is performed enzymatically, e.g., usingPoly(A) polymerase or any other enzyme suitable for catalyzing theincorporation of adenine residues at the 3′ terminus of the precursorRNA. Reaction mixtures for carrying out the adenylation reaction mayinclude any useful components, including but not limited to, apolymerase, a buffer (e.g., a Tris-HCL buffer), one or more metalcations (e.g., MgCl₂, MnCl₂, or combinations thereof), a salt (e.g.,NaCl), one or more enzyme-stabilizing components (e.g., DTT), ATP, andany other reaction components useful for facilitating the adenylation ofa precursor RNA. The adenylation reaction may be carried out at atemperature (e.g., 30° C.-50° C., such as 37° C.) and pH (e.g., pH 7-pH8.5, such as pH 7.9) compatible with the polymerase being employed,e.g., polyA polymerase. Other approaches for adding nucleotides to aprecursor RNA include ligation-based strategies, where an RNA ligase(e.g., T4 RNA ligase) catalyzes the covalent joining of a definedsequence to an end (e.g., the 3′ end) of the precursor RNA to producethe template RNA.

The methods of the present disclosure include combining a polymeraseinto the reaction mixture. A variety of polymerases may be employed whenpracticing the subject methods. The polymerase combined into thereaction mixture is capable of template switching, where the polymeraseuses a first nucleic acid strand as a template for polymerization, andthen switches to the 3′ end of a second “acceptor” template nucleic acidstrand to continue the same polymerization reaction. In certain aspects,the polymerase combined into the reaction mixture is a reversetranscriptase (RT). Reverse transcriptases capable of template-switchingthat find use in practicing the methods include, but are not limited to,retroviral reverse transcriptase, retrotransposon reverse transcriptase,retroplasmid reverse transcriptases, retron reverse transcriptases,bacterial reverse transcriptases, group II intron-derived reversetranscriptase, and mutants, variants derivatives, or functionalfragments thereof. For example, the reverse transcriptase may be aMoloney Murine Leukemia Virus reverse transcriptase (MMLV RT) or aBombyx mori reverse transcriptase (e.g., Bombyx mori R2 non-LTR elementreverse transcriptase). Polymerases capable of template switching thatfind use in practicing the subject methods are commercially availableand include SMARTScribe™ reverse transcriptase available from ClontechLaboratories, Inc. (Mountain View, Calif.). In certain aspects, a mix oftwo or more different polymerases is added to the reaction mixture,e.g., for improved processivity, proof-reading, and/or the like. In someinstances, the polymer is one that is heterologous relative to thetemplate, or source thereof.

The polymerase is combined into the reaction mixture such that the finalconcentration of the polymerase is sufficient to produce a desiredamount of the product nucleic acid. In certain aspects, the polymerase(e.g., a reverse transcriptase such as an MMLV RT or a Bombyx mori RT)is present in the reaction mixture at a final concentration of from 0.1to 200 units/μL (U/μL), such as from 0.5 to 100 U/μL, such as from 1 to50 U/μL, including from 5 to 25 U/μL, e.g., 20 U/μL.

In addition to a template switching capability, the polymerase combinedinto the reaction mixture may include other useful functionalities tofacilitate production of the product nucleic acid. For example, thepolymerase may have terminal transferase activity, where the polymeraseis capable of catalyzing template-independent addition ofdeoxyribonucleotides to the 3′ hydroxyl terminus of a DNA molecule. Incertain aspects, when the polymerase reaches the 5′ end of the templateRNA, the polymerase is capable of incorporating one or more additionalnucleotides at the 3′ end of the nascent strand not encoded by thetemplate. For example, when the polymerase has terminal transferaseactivity, the polymerase may be capable of incorporating 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more additional nucleotides at the 3′ end of thenascent DNA strand. In certain aspects, a polymerase having terminaltransferase activity incorporates 10 or less, such as 5 or less (e.g.,3) additional nucleotides at the 3′ end of the nascent DNA strand. Allof the nucleotides may be the same (e.g., creating a homonucleotidestretch at the 3′ end of the nascent strand) or at least one of thenucleotides may be different from the other(s). In certain aspects, theterminal transferase activity of the polymerase results in the additionof a homonucleotide stretch of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of thesame nucleotides (e.g., all dCTP, all dGTP, all dATP, or all dTTP).According to certain embodiments, the terminal transferase activity ofthe polymerase results in the addition of a homonucleotide stretch of 10or less, such as 9, 8, 7, 6, 5, 4, 3, or 2 (e.g., 3) of the samenucleotides. For example, according to one embodiment, the polymerase isan MMLV reverse transcriptase (MMLV RT). MMLV RT incorporates additionalnucleotides (predominantly dCTP, e.g., three dCTPs) at the 3′ end of thenascent DNA strand. As described in greater detail elsewhere herein,these additional nucleotides may be useful for enabling hybridizationbetween the 3′ end of the template switch oligonucleotide and the 3′ endof the nascent DNA strand, e.g., to facilitate template switching by thepolymerase from the template RNA to the template switch oligonucleotide.

As set forth above, the subject methods include combining a templateswitch nucleic acid into the reaction mixture. In certain aspects, thetemplate switch nucleic acid is a template switch oligonucleotide. By“template switch oligonucleotide” is meant an oligonucleotide templateto which a polymerase switches from an initial template (e.g., thetemplate RNA in the subject methods) during a nucleic acidpolymerization reaction. In this regard, the template RNA may bereferred to as a “donor template” and the template switcholigonucleotide may be referred to as an “acceptor template.” As usedherein, an “oligonucleotide” is a single-stranded multimer ofnucleotides from 2 to 500 nucleotides, e.g., 2 to 200 nucleotides.Oligonucleotides may be synthetic or may be made enzymatically, and, insome embodiments, are 10 to 50 nucleotides in length. Oligonucleotidesmay contain ribonucleotide monomers (i.e., may be oligoribonucleotidesor “RNA oligonucleotides”) or deoxyribonucleotide monomers (i.e., may beoligodeoxyribonucleotides or “DNA oligonucleotides”). Oligonucleotidesmay be 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to80, 80 to 100, 100 to 150 or 150 to 200, up to 500 or more nucleotidesin length, for example.

The reaction mixture includes the template switch oligonucleotide at aconcentration sufficient to permit template switching of the polymerasefrom the template RNA to the template switch oligonucleotide. Forexample, the template switch oligonucleotide may be added to thereaction mixture at a final concentration of from 0.01 to 100 μM, suchas from 0.1 to 10 μM, such as from 0.5 to 5 μM, including 1 to 2 μM(e.g., 1.2 μM).

The template switch oligonucleotide may include one or more nucleotides(or analogs thereof) that are modified or otherwise non-naturallyoccurring. For example, the template switch oligonucleotide may includeone or more nucleotide analogs (e.g., LNA, FANA, 2′-O-Me RNA, 2′-fluoroRNA, or the like), linkage modifications (e.g., phosphorothioates, 3′-3′and 5′-5′ reversed linkages), 5′ and/or 3′ end modifications (e.g., 5′and/or 3′ amino, biotin, DIG, phosphate, thiol, dyes, quenchers, etc.),one or more fluorescently labeled nucleotides, or any other feature thatprovides a desired functionality to the template switch oligonucleotide.

The template switch oligonucleotide includes a 3′ hybridization domainand a sequencing platform adapter construct. The 3′ hybridization domainmay vary in length, and in some instances ranges from 2 to 10 nts inlength, such as 3 to 7 nts in length. The sequence of the 3′hybridization may be any convenient sequence, e.g., an arbitrarysequence, a heterpolymeric sequence (e.g., a hetero-trinucleotide) orhomopolymeric sequence (e.g., a homo-trinucleotide, such as G-G-G), orthe like. Examples of 3′ hybridization domains and template switcholigonucleotides are further described in U.S. Pat. No. 5,962,272, thedisclosure of which is herein incorporated by reference. In addition toa 3′ hybridization domain, the template switch oligonucleotide includesa sequencing platform adapter construct. By “sequencing platform adapterconstruct” is meant a nucleic acid construct that includes at least aportion of a nucleic acid domain (e.g., a sequencing platform adapternucleic acid sequence) utilized by a sequencing platform of interest,such as a sequencing platform provided by Illumina® (e.g., the HiSeq™,MiSeq™ and/or Genome Analyzer™ sequencing systems); Ion Torrent™ (e.g.,the Ion PGM™ and/or Ion Proton™ sequencing systems); Pacific Biosciences(e.g., the PACBIO RS II sequencing system); Life Technologies™ (e.g., aSOLiD sequencing system); Roche (e.g., the 454 GS FLX+ and/or GS Juniorsequencing systems); or any other sequencing platform of interest.

In certain aspects, the sequencing platform adapter construct includes anucleic acid domain selected from: a domain (e.g., a “capture site” or“capture sequence”) that specifically binds to a surface-attachedsequencing platform oligonucleotide (e.g., the P5 or P7 oligonucleotidesattached to the surface of a flow cell in an Illumina® sequencingsystem); a sequencing primer binding domain (e.g., a domain to which theRead 1 or Read 2 primers of the Illumina® platform may bind); a barcodedomain (e.g., a domain that uniquely identifies the sample source of thenucleic acid being sequenced to enable sample multiplexing by markingevery molecule from a given sample with a specific barcode or “tag”); abarcode sequencing primer binding domain (a domain to which a primerused for sequencing a barcode binds); a molecular identification domain(e.g., a molecular index tag, such as a randomized tag of 4, 6, or othernumber of nucleotides) for uniquely marking molecules of interest todetermine expression levels based on the number of instances a uniquetag is sequenced; or any combination of such domains. In certainaspects, a barcode domain (e.g., sample index tag) and a molecularidentification domain (e.g., a molecular index tag) may be included inthe same nucleic acid.

The sequencing platform adapter constructs may include nucleic aciddomains (e.g., “sequencing adapters”) of any length and sequencesuitable for the sequencing platform of interest. In certain aspects,the nucleic acid domains are from 4 to 200 nucleotides in length. Forexample, the nucleic acid domains may be from 4 to 100 nucleotides inlength, such as from 6 to 75, from 8 to 50, or from 10 to 40 nucleotidesin length. According to certain embodiments, the sequencing platformadapter construct includes a nucleic acid domain that is from 2 to 8nucleotides in length, such as from 9 to 15, from 16-22, from 23-29, orfrom 30-36 nucleotides in length.

The nucleic acid domains may have a length and sequence that enables apolynucleotide (e.g., an oligonucleotide) employed by the sequencingplatform of interest to specifically bind to the nucleic acid domain,e.g., for solid phase amplification and/or sequencing by synthesis ofthe cDNA insert flanked by the nucleic acid domains. Example nucleicacid domains include the P5 (5′-AATGATACGGCGACCACCGA-3′) (SEQ ID NO:01),P7 (5′-CAAGCAGAAGACGGCATACGAGAT-3′) (SEQ ID NO:02), Read 1 primer(5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′) (SEQ ID NO:03) and Read 2primer (5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′) (SEQ ID NO:04) domainsemployed on the Illumina®-based sequencing platforms. Other examplenucleic acid domains include the A adapter(5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′) (SEQ ID NO:05) and P1 adapter(5′-CCTCTCTATGGGCAGTCGGTGAT-3′) (SEQ ID NO:06) domains employed on theIon Torrent™-based sequencing platforms.

The nucleotide sequences of nucleic acid domains useful for sequencingon a sequencing platform of interest may vary and/or change over time.Adapter sequences are typically provided by the manufacturer of thesequencing platform (e.g., in technical documents provided with thesequencing system and/or available on the manufacturer's website). Basedon such information, the sequence of the sequencing platform adapterconstruct of the template switch oligonucleotide (and optionally, afirst strand synthesis primer, amplification primers, and/or the like)may be designed to include all or a portion of one or more nucleic aciddomains in a configuration that enables sequencing the nucleic acidinsert (corresponding to the template RNA) on the platform of interest.

According to certain embodiments, the template switch oligonucleotideincludes a modification that prevents the polymerase from switching fromthe template switch oligonucleotide to a different template nucleic acidafter synthesizing the compliment of the 5′ end of the template switcholigonucleotide (e.g., a 5′ adapter sequence of the template switcholigonucleotide). Useful modifications include, but are not limited to,an abasic lesion (e.g., a tetrahydrofuran derivative), a nucleotideadduct, an iso-nucleotide base (e.g., isocytosine, isoguanine, and/orthe like), and any combination thereof.

The template switch oligonucleotide may include a sequence (e.g., adefined nucleotide sequence 5′ of the 3′ hybridization domain of thetemplate switch oligonucleotide), that enables second strand synthesisand/or PCR amplification of the single product nucleic acid. Forexample, the template switch oligonucleotide may include a sequence,where subsequent to generating the single product nucleic acid, secondstrand synthesis is performed using a primer that has that sequence. Thesecond strand synthesis produces a second strand DNA complementary tothe single product nucleic acid. Alternatively, or additionally, thesingle product nucleic acid may be amplified using a primer pair inwhich one of the primers has that sequence. Accordingly, in certainaspects, the methods of the present disclosure may further includeproducing the product nucleic acid and contacting a 3′ region of thesingle product nucleic acid complementary to the template switcholigonucleotide with a second strand primer configured to bind theretounder hybridization conditions. Following contacting the 3′ region ofthe single product nucleic acid complementary to the template switcholigonucleotide with the second strand primer, the methods may furtherinclude subjecting the reaction mixture to nucleic acid polymerizationconditions.

The term “complementary” as used herein refers to a nucleotide sequencethat base-pairs by non-covalent bonds to all or a region of a targetnucleic acid (e.g., a region of the product nucleic acid). In thecanonical Watson-Crick base pairing, adenine (A) forms a base pair withthymine (T), as does guanine (G) with cytosine (C) in DNA. In RNA,thymine is replaced by uracil (U). As such, A is complementary to T andG is complementary to C. In RNA, A is complementary to U and vice versa.Typically, “complementary” refers to a nucleotide sequence that is atleast partially complementary. The term “complementary” may alsoencompass duplexes that are fully complementary such that everynucleotide in one strand is complementary to every nucleotide in theother strand in corresponding positions. In certain cases, a nucleotidesequence may be partially complementary to a target, in which not allnucleotides are complementary to every nucleotide in the target nucleicacid in all the corresponding positions. For example, a primer may beperfectly (i.e., 100%) complementary to the target nucleic acid, or theprimer and the target nucleic acid may share some degree ofcomplementarity which is less than perfect (e.g., 70%, 75%, 85%, 90%,95%, 99%). The percent identity of two nucleotide sequences can bedetermined by aligning the sequences for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first sequence foroptimal alignment). The nucleotides at corresponding positions are thencompared, and the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity=# of identical positions/total # of positions×100).When a position in one sequence is occupied by the same nucleotide asthe corresponding position in the other sequence, then the molecules areidentical at that position. A non-limiting example of such amathematical algorithm is described in Karlin et al., Proc. Natl. Acad.Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) as described in Altschul etal., Nucleic Acids Res. 25:389-3402 (1997). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., NBLAST) can be used. In one aspect, parameters for sequencecomparison can be set at score=100, wordlength=12, or can be varied(e.g., wordlength=5 or wordlength=20).

As used herein, the term “hybridization conditions” means conditions inwhich a primer specifically hybridizes to a region of the target nucleicacid (e.g., the template RNA, the single product nucleic acid, etc.).Whether a primer specifically hybridizes to a target nucleic acid isdetermined by such factors as the degree of complementarity between thepolymer and the target nucleic acid and the temperature at which thehybridization occurs, which may be informed by the melting temperature(T_(M)) of the primer. The melting temperature refers to the temperatureat which half of the primer-target nucleic acid duplexes remainhybridized and half of the duplexes dissociate into single strands. TheT_(m) of a duplex may be experimentally determined or predicted usingthe following formula T_(m)=81.5+16.6(log₁₀[Na⁺])+0.41 (fractionG+C)−(60/N), where N is the chain length and [Na⁺] is less than 1 M. SeeSambrook and Russell (2001; Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor N.Y., Ch. 10).Other more advanced models that depend on various parameters may also beused to predict T_(m) of primer/target duplexes depending on varioushybridization conditions. Approaches for achieving specific nucleic acidhybridization may be found in, e.g., Tijssen, Laboratory Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” Elsevier (1993).

As described above, the subject methods include combining dNTPs into thereaction mixture. In certain aspects, each of the fournaturally-occurring dNTPs (dATP, dGTP, dCTP and dTTP) are added to thereaction mixture. For example, dATP, dGTP, dCTP and dTTP may be added tothe reaction mixture such that the final concentration of each dNTP isfrom 0.01 to 100 mM, such as from 0.1 to 10 mM, including 0.5 to 5 mM(e.g., 1 mM). According to one embodiment, at least one type ofnucleotide added to the reaction mixture is a non-naturally occurringnucleotide, e.g., a modified nucleotide having a binding or other moiety(e.g., a fluorescent moiety) attached thereto, a nucleotide analog, orany other type of non-naturally occurring nucleotide that finds use inthe subject methods or a downstream application of interest.

The addition of a primer to the reaction mixture is not necessary whenthe template RNA provides a suitable substrate for initiation offirst-strand synthesis. For example, when the template RNA hasdouble-stranded regions and an overhang at one or both of its ends, the“non-overhanging” strand of the dsRNA can prime a first-strand synthesisreaction in which the overhanging strand serves as the template. In thismanner, the polymerase may be used to “fill in” the overhang, switch tothe template switch oligonucleotide, and complete the first strandsynthesis using the template switch oligonucleotide as an acceptortemplate to produce the product nucleic acid (where a terminaltransferase reaction by the polymerase optionally precedes the templateswitch as described elsewhere herein). Accordingly, the addition of aprimer is obviated when the template RNA includes, e.g., an overhang atone or both of its ends.

In certain circumstances, however, it may be desirable to add a primerto the reaction mixture to prime the synthesis of the single productnucleic acid. For example, if the template RNA is single-stranded, aprimer may be useful for purposes of initiating first-strand synthesis.In addition, use of a primer can give a practitioner of the subjectmethods more control over which RNA(s) in an RNA sample will serve asthe template RNA(s) for production of the product nucleic acid, e.g.,where it is desirable to produce product nucleic acids corresponding toa template RNA of interest (e.g., polyadenylated RNA, for which an oligodT-based primer that hybridizes to the polyA tail of the RNA may be usedto prime the first strand synthesis).

Accordingly, in certain aspects, the subject methods further includecontacting the template RNA with a first primer that primes thesynthesis of the single product nucleic acid. The contacting isperformed under conditions sufficient for the primer to hybridize to thetemplate RNA, which conditions are described elsewhere herein. Accordingto one embodiment, the entire sequence of the primer is arbitrary, e.g.,the primer may be a random hexamer or any other random primer ofsuitable length (or mixtures thereof). In other aspects, the primer hasa defined sequence, e.g., the primer sequence may be designed by onepracticing the subject methods to specifically hybridize to a knowncomplementary sequence in a template RNA of interest (e.g., a polyA tailof the template RNA).

According to certain embodiments, the primer includes two or moredomains. For example, the primer may include a first (e.g., 3′) domainthat hybridizes to the template RNA and a second (e.g., 5′) domain thatdoes not hybridize to the template RNA. The sequence of the first andsecond domains may be independently defined or arbitrary. In certainaspects, the first domain has a defined sequence and the sequence of thesecond domain is defined or arbitrary. In other aspects, the firstdomain has an arbitrary sequence (e.g., a random sequence, such as arandom hexamer sequence) and the sequence of the second domain isdefined or arbitrary. According to one embodiment, the second domainincludes a nucleotide sequence that is the same as, or different from, anucleotide sequence present in the template switch oligonucleotide.

In some embodiments, the second domain of the primer includes asequencing platform adapter construct. The sequencing platform adapterconstruct of the second domain may include a nucleic acid domainselected from a domain (e.g., a “capture site” or “capture sequence”)that specifically binds to a surface-attached sequencing platformoligonucleotide (e.g., the P5 or P7 oligonucleotides attached to thesurface of a flow cell in an Illumina® sequencing system), a sequencingprimer binding domain (e.g., a domain to which the Read 1 or Read 2primers of the Illumina® platform may bind), a barcode domain (e.g., adomain that uniquely identifies the sample source of the nucleic acidbeing sequenced to enable sample multiplexing by marking every moleculefrom a given sample with a specific barcode or “tag”), a barcodesequencing primer binding domain (a domain to which a primer used forsequencing a barcode binds), a molecular identification domain, or anycombination of such domains.

In certain aspects, the sequencing platform adapter construct of thesecond domain of the primer is different from the sequencing platformadapter construct of the template switch oligonucleotide. Suchembodiments find use, e.g., where one wishes to produce a single productnucleic acid (e.g., a cDNA or library thereof) with one end having oneor more sequencing platform adapter sequences and the second end havingone or more sequencing platform adapter sequences different from thefirst end. Having ends with different adapter sequences is useful, e.g.,for subsequent solid phase amplification (e.g., cluster generation usingthe surface-attached P5 and P7 primers in an Illumina®-based sequencingsystem), DNA sequencing (e.g., using the Read 1 and Read 2 primers in anIllumina®-based sequencing system), and any other steps performed by asequencing platform requiring different adapter sequences at opposingends of the nucleic acid to be sequenced. Having different ends is alsouseful in providing strand specific information, since thedirectionality of the sequenced strand is defined by the different ends.Current methods in the art for doing this require multiple steps anddegradation of the undesired strand—e.g., using UDG and incorporation ofdU into the undesired strand. The current method is far more streamlinedand requires less steps, generating strand-specific informationdirectly.

When the methods include contacting the template RNA with a primer thatprimes the synthesis of the single product nucleic acid, the primer mayinclude one or more nucleotides (or analogs thereof) that are modifiedor otherwise non-naturally occurring. For example, the primer mayinclude one or more nucleotide analogs (e.g., LNA, FANA, 2′-O-Me RNA,2′-fluoro RNA, or the like), linkage modifications (e.g.,phosphorothioates, 3′-3′ and 5′-5′ reversed linkages), 5′ and/or 3′ endmodifications (e.g., 5′ and/or 3′ amino, biotin, DIG, phosphate, thiol,dyes, quenchers, etc.), one or more fluorescently labeled nucleotides,or any other feature that provides a desired functionality to the primerthat primes the synthesis of the single product nucleic acid.

In certain aspects, when the methods include contacting the template RNAwith a primer that primes the synthesis of the single product nucleicacid, it may be desirable to prevent any subsequent extension reactionswhich use the single product nucleic acid as a template from extendingbeyond a particular position in the region of the single product nucleicacid corresponding to the primer. For example, according to certainembodiments, the primer that primes the synthesis of the single productnucleic acid includes a modification that prevents a polymerase usingthe region corresponding to the primer as a template from polymerizing anascent strand beyond the modification. Useful modifications include,but are not limited to, an abasic lesion (e.g., a tetrahydrofuranderivative), a nucleotide adduct, an iso-nucleotide base (e.g.,isocytosine, isoguanine, and/or the like), and any combination thereof.

Any nucleic acids that find use in practicing the methods of the presentdisclosure (e.g., the template switch oligonucleotide, a primer thatprimes the synthesis of the single product nucleic acid, a second strandsynthesis primer, one or more primers for amplifying the product nucleicacid, and/or the like) may include any useful nucleotide analoguesand/or modifications, including any of the nucleotide analogues and/ormodifications described herein.

Once the product nucleic acid is produced, the methods may includeinputting the product nucleic acid directly into a downstreamapplication of interest (e.g., a sequencing application, etc.). In otheraspects, the methods may include using the product nucleic acid as atemplate for second-strand synthesis and/or PCR amplification (e.g., forsubsequent sequencing of the amplicons). According to one embodiment,the methods of the present disclosure further include subjecting theproduct nucleic acid to nucleic acid amplification conditions. Suchconditions may include the addition of forward and reverse primersconfigured to amplify all or a desired portion of the product nucleicacid, dNTPs, and a polymerase suitable for effecting the amplification(e.g., a thermostable polymerase). The single product nucleic acid mayhave an amplification sequence at its 5′ end and an amplificationsequence at its 3′ end, and be subjected to PCR amplification conditionswith primers complementary to the 5′ and 3′ amplification sequences. Theamplification sequences may be (or overlap with) a nucleic acid domainin a sequencing platform adapter construct, or may be outside of thesequencing platform adapter construct. An initial step in carrying outthe amplification may include denaturing the product nucleic acid todissociate the template RNA and template switch oligonucleotide from thesingle product nucleic acid, thereby making the single product nucleicacid available for primer binding.

In certain aspects, when the single product nucleic acid is amplifiedfollowing its production, the amplification may be carried out using aprimer pair in which one or both of the primers include a sequencingplatform adapter construct. The sequencing platform adapter construct(s)may include any of the nucleic acid domains described elsewhere herein(e.g., a domain that specifically binds to a surface-attached sequencingplatform oligonucleotide, a sequencing primer binding domain, a barcodedomain, a barcode sequencing primer binding domain, a molecularidentification domain, or any combination thereof). Such embodimentsfinds use, e.g., where the single product nucleic does not include allof the adapter domains useful or necessary for sequencing in asequencing platform of interest, and the remaining adapter domains areprovided by the primers used for the amplification of the single productnucleic acid. An example method according to this embodiment is shown inFIG. 1. As shown, template RNA 102, polymerase 104, template switcholigonucleotide 106, and dNTPs (not shown) are combined into reactionmixture 100 under conditions sufficient to produce the product nucleicacid. Template switch oligonucleotide 106 includes sequencing platformadapter construct B. Although optional, the embodiment shown in FIG. 1employs a first primer, primer 108, which is extended by the polymerasefor first strand synthesis. Primer 108 includes first (3′) domain 110that hybridizes to the template RNA and second (5′) domain 112 that doesnot hybridize to the template RNA. The second domain includes sequencingplatform adapter construct A. The nucleotide sequence of first domain110 may be arbitrary (e.g., a random sequence, such as a random hexamersequence) or the sequence of the first domain may be defined (e.g., asequence specifically selected to hybridize to a particular region of aparticular template RNA of interest). In this example, first domain 110of primer 108 is complementary to sequence 114 within template RNA 102,and second domain 112 includes sequencing platform adapter construct Ahaving one or more sequencing platform nucleic acid domains (e.g., adomain that specifically binds to a surface-attached sequencing platformoligonucleotide, a sequencing primer binding domain, a barcode domain, abarcode sequencing primer binding domain, a molecular identificationdomain, and combinations thereof).

Upon hybridization of primer 108 to template RNA 102, first strandsynthesis proceeds when polymerase 104 extends primer 108 along templateRNA 102. In this example, the polymerase has terminal transferaseactivity, such that when the extension reaction reaches the 5′ end ofthe template RNA, the polymerase adds an arbitrary sequence that can behomodimeric or heterodimeric, and may range in length of nucleotides(e.g., 2 to 10 nts, such as 2 to 5 nts) such as a homonucleotide stretch(e.g., a homo-trinucleotide shown here as NNN) to the extension product.According to this embodiment, template switch oligonucleotide has a 3′hybridization domain that includes a homonucleotide stretch (shown hereas a homo-trinucleotide stretch, NNN) complementary to thehomonucleotide stretch at the 3′ end of the extension product. Thiscomplementarity promotes hybridization of the 3′ hybridization domain ofthe template switch oligonucleotide to the 3′ end of the extensionproduct. Hybridization brings the acceptor template region of thetemplate switch oligonucleotide (located 5′ of the 3′ hybridizationdomain) within sufficient proximity of the polymerase such that thepolymerase can template switch to the acceptor template region andcontinue the extension reaction to the 5′ terminal nucleotide of thetemplate switch oligonucleotide, thereby producing the product nucleicacid that includes the template RNA and the template switcholigonucleotide each hybridized to adjacent regions of the singleproduct nucleic acid.

In this example, the template switch oligonucleotide includes sequencingplatform adapter construct B having one or more sequencing platformnucleic acid domains (e.g., a domain that specifically binds to asurface-attached sequencing platform oligonucleotide, a sequencingprimer binding domain, a barcode domain, a barcode sequencing primerbinding domain, a molecular identification domain, and combinationsthereof), such that the single product nucleic acid includes sequencingplatform adapter construct A at its 5′ end and sequencing platformadapter construct B′ at its 3′ end. According to this embodiment, themethod further includes a second strand synthesis step, where a primercomplementary to a 3′ region of the single product nucleic acidhybridizes to the 3′ region of the single product nucleic acid and isextended by a polymerase—using the single product nucleic acid as atemplate—to the 5′ end of the single product nucleic acid. The result ofthis second strand synthesis step is a double-stranded DNA that includesthe single product nucleic acid and its complementary strand.

In the example shown in FIG. 1, adapter constructs A/A′ and B/B′ do notinclude all of the sequencing platform nucleic acid domains useful ornecessary for downstream sequencing of the nucleic acid. To add theremaining sequencing platform nucleic acid domains, the nucleic acid isamplified using primers having adapter constructs C and D (e.g., presentin a non-hybridizing 5′ region of the primers) which provide theremaining sequencing platform nucleic acid domains. The ampliconsinclude adapter constructs A/A′ and C/C′ at one end and adapterconstructs B/B′ and D/D′ at the opposite end. One practicing the subjectmethods may select the sequences of the sequencing platform adapterconstruct of the first strand synthesis primer, the template switcholigonucleotide, and the amplification primers, to provide all of thenecessary domains in a suitable configuration for sequencing on asequencing platform of interest. As just one example, constructs A/A′and B/B′ may include sequencing primer binding domains (e.g., primerbinding domains for the Read 1 and Read 2 sequencing primers employed inIllumina®-based sequencing platforms), while constructs C/C′ and D/D′include a domain that specifically binds to a surface-attachedsequencing platform oligonucleotide (e.g., domains that specificallybind to the surface-attached P5 and P7 primers of an Illumina®sequencing system). Any of adapter constructs A/A′-D/D′ may include anyadditional sequence elements useful or necessary for sequencing on asequencing platform of interest.

As summarize above, a primer having a sequencing platform adapterconstruct may be used to prime the synthesis of the single productnucleic acid, so that the single product nucleic acid has a sequencingplatform adapter construct at its 5′ and 3′ ends. In certain aspects,the sequencing platform adapter constructs of the single product nucleicacid include all of the useful or necessary domains for sequencing thenucleic acid on a sequencing platform of interest. As shown in FIG. 2, aproduct nucleic acid is produced using an approach similar to that shownin FIG. 1. However, in the embodiment shown in FIG. 2, sequencingadapter constructs A/A′ and B/B′ include all of the sequencing platformnucleic acid domains useful or necessary for sequencing the singleproduct nucleic acid on a sequencing platform of interest (e.g., adomain that specifically binds to a surface-attached sequencing platformoligonucleotide, a sequencing primer binding domain, a barcode domain, abarcode sequencing primer binding domain, a molecular identificationdomain, and combinations thereof). According to certain embodiments, thesingle product nucleic acid is PCR amplified prior to sequencing on thesequencing platform. In other embodiments, the single product nucleicacid is not amplified prior to sequencing.

A method according to an additional embodiment of the present disclosureis shown in FIG. 3. In this example, non-polyadenylated precursor RNA302 undergoes 3′ polyadenylation to produce template RNA 303. In thisexample, first strand synthesis is primed using an oligo(dT) primerhaving a sequencing platform adapter construct (A) at its 5′ end, sothat the single product nucleic acid has sequencing platform adapterconstructs A and B′ at its 5′ and 3′ ends, respectively. The sequencingplatform adapter constructs may include less than all of the useful ornecessary domains for sequencing on a sequencing platform of interest(e.g., similar to the embodiment shown in FIG. 1) or may include alluseful or necessary domains (e.g., similar to the embodiment shown inFIG. 2). Embodiments such as the one shown in FIG. 3 find use, e.g., ingenerating a sequencing-ready library of cDNAs which correspond tonon-polyadenylated RNAs (e.g., microRNAs, small RNAs, siRNAs, or thelike) present in a biological sample of interest.

In certain embodiments, the subject methods may be used to generate acDNA library corresponding to mRNAs for downstream sequencing on asequencing platform of interest (e.g., a sequencing platform provided byIllumina®, Ion Torrent™, Pacific Biosciences, Life Technologies™, Roche,or the like). In one embodiment, mRNAs are sheared to a length ofapproximately 200 bp, or any other appropriate length as defined by thesequencing platform being used (e.g. 400-800 bp), and then used astemplates in a template switch polymerization reaction as describedelsewhere herein. The first strand synthesis is primed using a primerhaving a sequencing primer binding domain (e.g., an Illumina® Read 2 N6primer binding domain), and the template switch oligonucleotide includesa second sequencing primer binding domain of the sequencing platform(e.g., an Illumina® Read 1 primer binding domain). In certain aspects,the first strand synthesis is primed using a random primer. Theresulting library may then optionally be PCR amplified with primers thatadd nucleic acid domains that bind to surface-attached sequencingplatform oligonucleotides (e.g., the P5 and P7 oligonucleotides attachedto the flow cell in an Illumina® sequencing system). The library may bemixed 50:50 with a control library (e.g., Illumina®'s PhiX controllibrary) and sequenced on the sequencing platform (e.g., an Illumina®sequencing system). The control library sequences may be removed and theremaining sequences mapped to the transcriptome of the source of themRNAs (e.g., human, mouse, or any other mRNA source).

According to certain embodiments, the subject methods may be used togenerate a cDNA library corresponding to non-polyadenylated RNAs fordownstream sequencing on an Illumina®-based sequencing system. In oneembodiment, microRNAs are polyadenylated and then used as templates in atemplate switch polymerization reaction as described elsewhere herein.The first strand synthesis is primed using an Illumina® dT primer, andthe template switch oligonucleotide included an Illumina® Read 1 primerbinding domain.

FIG. 5 shows example sequences that may be added to nucleic acidsaccording to one embodiment of the present disclosure. In this example,a template switch oligonucleotide (top) includes a 3′ hybridizationdomain (GGG) and a sequencing platform adapter construct that includes abinding site for a surface-attached sequencing platform oligonucleotide(in this example, the surface-attached P5 primer of an Illumina® system)and a sequencing primer binding site (in this example, a binding sitefor the Read 1 sequencing primer of an Illumina® system) to facilitatesequencing on a sequencing platform of interest. A sequencing platformadapter construct (bottom) which may be included in the nucleic acid atan end opposite the template switch oligonucleotide includes a bindingsite for a second surface-attached sequencing platform oligonucleotide(in this example, the surface-attached P7 primer of an Illumina®system), an index barcode, and a second sequencing primer binding site(in this example, the binding site for a Read 2 sequencing primer of anIllumina® system) to facilitate sequencing on a sequencing platform ofinterest.

The subject methods may further include combining a thermostablepolymerase (e.g., a Taq, Pfu, Tfl, Tth, Tli, and/or other thermostablepolymerase)—in addition to the template switching polymerase—into thereaction mixture. Alternatively, the template switching polymerase maybe a thermostable polymerase. Either of these embodiments find use,e.g., when it is desirable to achieve sequencing platform adapterconstruct addition and amplification (e.g., amplification with orwithout further adapter addition) of the product nucleic acid in asingle tube. For example, the contents of the single tube may be placedunder conditions suitable for the template switch polymerizationreaction to occur (as described elsewhere herein), followed by placingthe reaction contents under thermocycling conditions (e.g.,denaturation, primer annealing, and polymerization conditions) in whichthe first-strand synthesis product is PCR amplified using amplificationprimers and the thermostable polymerase present in the single tube. Dueto its thermostability, the thermostable polymerase will retain itsactivity even when present during the PCR phase of this embodiment.

Compositions

Also provided by the present disclosure are compositions. The subjectcompositions may include, e.g., one or more of any of the reactionmixture components described above with respect to the subject methods.For example, the compositions may include one or more of a templateribonucleic acid (RNA), a polymerase (e.g., a polymerase capable oftemplate-switching, a thermostable polymerase, combinations thereof, orthe like), a template switch oligonucleotide, dNTPs, a salt, a metalcofactor, one or more nuclease inhibitors (e.g., an RNase inhibitor),one or more enzyme-stabilizing components (e.g., DTT), or any otherdesired reaction mixture component(s).

In certain aspects, the subject compositions include a templateribonucleic acid (RNA) and a template switch oligonucleotide eachhybridized to adjacent regions of a nucleic acid strand, where thetemplate switch oligonucleotide includes a 3′ hybridization domain and asequencing platform adapter construct. The sequencing platform adapterconstruct may include any sequencing platform nucleic acid domain ofinterest, including any of the domains described above with respect tothe subject methods (e.g., a domain that specifically binds to asurface-attached sequencing platform oligonucleotide, a sequencingprimer binding domain, a barcode domain, a barcode sequencing primerbinding domain, a molecular identification domain, or any combinationthereof). Approaches for isolating RNA samples from a nucleic acidsource of interest, as well as strategies for generating template RNAsfrom precursor RNAs, are described elsewhere herein.

In certain aspects, the 3′ hybridization domain of the template switcholigonucleotide includes an arbitrary sequence, e.g., as describedabove.

The subject compositions may be present in any suitable environment.According to one embodiment, the composition is present in a reactiontube (e.g., a 0.2 mL tube, a 0.6 mL tube, a 1.5 mL tube, or the like) ora well. In certain aspects, the composition is present in two or more(e.g., a plurality of) reaction tubes or wells (e.g., a plate, such as a96-well plate). The tubes and/or plates may be made of any suitablematerial, e.g., polypropylene, or the like. In certain aspects, thetubes and/or plates in which the composition is present provide forefficient heat transfer to the composition (e.g., when placed in a heatblock, water bath, thermocycler, and/or the like), so that thetemperature of the composition may be altered within a short period oftime, e.g., as necessary for a particular enzymatic reaction to occur.According to certain embodiments, the composition is present in athin-walled polypropylene tube, or a plate having thin-walledpolypropylene wells. In certain embodiments it may be convenient for thereaction to take place on a solid surface or a bead, in such case, thetemplate switch oligonucleotide or one or more of the primers may beattached to the solid support or bead by methods known in the art—suchas biotin linkage or by covalent linkage) and reaction allowed toproceed on the support.

Other suitable environments for the subject compositions include, e.g.,a microfluidic chip (e.g., a “lab-on-a-chip device”). The compositionmay be present in an instrument configured to bring the composition to adesired temperature, e.g., a temperature-controlled water bath, heatblock, or the like. The instrument configured to bring the compositionto a desired temperature may be configured to bring the composition to aseries of different desired temperatures, each for a suitable period oftime (e.g., the instrument may be a thermocycler).

Kits

Aspects of the present disclosure also include kits. The kits mayinclude, e.g., one or more of any of the reaction mixture componentsdescribed above with respect to the subject methods. For example, thekits may include one or more of a template ribonucleic acid (RNA),components for producing a template RNA from a precursor RNA (e.g., apoly(A) polymerase and associated reagents for polyadenylating anon-polyadenylated precursor RNA), a polymerase (e.g., a polymerasecapable of template-switching, a thermostable polymerase, combinationsthereof, or the like), a template switch oligonucleotide, dNTPs, a salt,a metal cofactor, one or more nuclease inhibitors (e.g., an RNaseinhibitor and/or a DNase inhibitor), one or more molecular crowdingagents (e.g., polyethylene glycol, or the like), one or moreenzyme-stabilizing components (e.g., DTT), or any other desired kitcomponent(s), such as solid supports, e.g., tubes, beads, microfluidicchips, etc.

According to one embodiment, the subject kits include a template switcholigonucleotide comprising a 3′ hybridization domain and a sequencingplatform adapter construct, and a template switching polymerase. Thesequencing platform adapter construct may include any sequencingplatform nucleic acid domain of interest, including any of the domainsdescribed above with respect to the subject methods and compositions(e.g., a domain that specifically binds to a surface-attached sequencingplatform oligonucleotide, a sequencing primer binding domain, a barcodedomain, a barcode sequencing primer binding domain, a molecularidentification domain, or any combination thereof).

Kits of the present disclosure may include a first-strand synthesisprimer that includes a first domain that hybridizes to a template RNAand a second domain that does not hybridize to the template RNA. Thefirst domain may have a defined or arbitrary sequence. The second domainof such primers may include, e.g., a sequencing platform adapterconstruct that includes a nucleic acid domain selected from a domainthat specifically binds to a surface-attached sequencing platformoligonucleotide, a sequencing primer binding domain, a barcode domain, abarcode sequencing primer binding domain, a molecular identificationdomain, and any combination thereof.

In certain embodiments, the kits include reagents for isolating RNA froma source of RNA. The reagents may be suitable for isolating nucleic acidsamples from a variety of RNA sources including single cells, culturedcells, tissues, organs, or organisms. The subject kits may includereagents for isolating a nucleic acid sample from a fixed cell, tissueor organ, e.g., formalin-fixed, paraffin-embedded (FFPE) tissue. Suchkits may include one or more deparaffinization agents, one or moreagents suitable to de-crosslink nucleic acids, and/or the like.

Components of the kits may be present in separate containers, ormultiple components may be present in a single container. For example,the template switch oligonucleotide and the template switchingpolymerase may be provided in the same tube, or may be provided indifferent tubes. In certain embodiments, it may be convenient to providethe components in a lyophilized form, so that they are ready to use andcan be stored conveniently at room temperature.

In addition to the above-mentioned components, a subject kit may furtherinclude instructions for using the components of the kit, e.g., topractice the subject method. The instructions are generally recorded ona suitable recording medium. For example, the instructions may beprinted on a substrate, such as paper or plastic, etc. As such, theinstructions may be present in the kits as a package insert, in thelabeling of the container of the kit or components thereof (i.e.,associated with the packaging or subpackaging) etc. In otherembodiments, the instructions are present as an electronic storage datafile present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, Hard Disk Drive (HDD) etc. In yet other embodiments,the actual instructions are not present in the kit, but means forobtaining the instructions from a remote source, e.g. via the internet,are provided. An example of this embodiment is a kit that includes a webaddress where the instructions can be viewed and/or from which theinstructions can be downloaded. As with the instructions, this means forobtaining the instructions is recorded on a suitable substrate.

Utility

The subject methods find use in a variety of applications, includingthose that require the presence of particular nucleotide sequences atone or both ends of nucleic acids of interest. Such applications existin the areas of basic research and diagnostics (e.g., clinicaldiagnostics) and include, but are not limited to, the generation ofsequencing-ready cDNA libraries. Such libraries may include adaptersequences that enable sequencing of the library members using anyconvenient sequencing platform, including: the HiSeq™, MiSeq™ and GenomeAnalyzer™ sequencing systems from Illumina®; the Ion PGM™ and IonProton™ sequencing systems from Ion Torrent™; the PACBIO RS IIsequencing system from Pacific Biosciences, the SOLiD sequencing systemsfrom Life Technologies™, the 454 GS FLX+ and GS Junior sequencingsystems from Roche, or any other convenient sequencing platform. Themethods of the present disclosure find use in generating sequencingready cDNA libraries corresponding to any RNA starting material ofinterest (e.g., mRNA) and are not limited to polyadenylated RNAs. Forexample, the subject methods may be used to generate sequencing-readycDNA libraries from non-polyadenylated RNAs, including microRNAs, smallRNAs, siRNAs, and/or any other type non-polyadenylated RNAs of interest.The methods also find use in generating strand-specific information,which can be helpful in determining allele-specific expression or indistinguishing overlapping transcripts in the genome.

An aspect of the subject methods is that—utilizing a template RNA—a cDNAspecies having sequencing platform adapter sequences at one or both ofits ends is generated in a single step, e.g., without the added stepsassociated with traditional ligation-based approaches for generatinghybrid nucleic acid molecules for downstream sequencing applications.Such steps include a ligation step (which may require a priorrestriction digest), washing steps, and any other necessary stepsassociated with traditional ligation-based approaches. Accordingly, themethods of the present disclosure are more efficient, cost-effective,and provide more flexibility than the traditional approaches.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental I. Library Construction

1 μg of Human Brain PolyA RNA (Clontech) was fragmented with addition of5× fragmentation Buffer (200 mM Tris acetate, pH 8.2, 500 mM potassiumacetate, and 150 mM magnesium acetate) and heating at 94° C. for 2 min30 s. Fragmented RNA was purified using a Nucleospin RNA XS spin column(Macharey Nagel).

Fragmented RNA was diluted to either 1 ng/μl of 5 ng/μl in RNase freewater. 1 μl of fragmented RNA or water was combined with 1 μl 120 firststrand primer and 2.5 μl of RNase free water. Samples were heated to 72°C. for 3 minutes and then placed on ice. To these samples were added 2μl 5× first strand buffer (Clontech), 0.25 μl 100 mM DTT, 0.25 μlrecombinant RNase inhibitor (Takara), 10 mM dNTP mix (Clontech), 1 μl120 template switch oligo and 1 μl SMARTScribe RT (Clontech). Sampleswere incubated at 42° C. for 90 minutes followed by 70° C. for 10minutes.

First strand cDNA reactions were purified by addition of 15 μl water and25 μl Ampure XP beads (Beckman Coulter). Samples were well mixed andincubated at room temperature for 8 minutes. Samples were bound to amagnetic stand for 5 minutes, and the beads were washed twice with 200μl 80% ethanol and allowed to air dry for 5 minutes.

cDNA on the beads was eluted by addition of 50 μl PCR Mastermix (5 μl 10Advantage2 buffer, 5 μl GC Melt reagent, 1 μl 10 mM dNTPs, 1 μlAdvantage2 polymerase (Clontech), 240 nM forward PCR primer, 240 nMreverse PCR primer, and 36.8 μl water). Samples were thermocycled for 12PCR cycles with the settings 95° C. 1 minute, 12× (95° C. 15 seconds,65° C. 30 seconds, 68° C. 1 minute). PCR products were purified with 50μl Ampure XP beads and eluted in 40 μl TE buffer.

Samples were diluted and run on an Agilent Bioanalyzer using the highsensitivity DNA assay. The results are provided in FIG. 4.

II. Construction of Illumina Sequenced Libraries A. Library Construction

1 μg of Mouse Brain PolyA RNA (Clontech) was fragmented addition of 5×fragmentation Buffer (200 mM Tris acetate, pH 8.2, 500 mM potassiumacetate, and 150 mM magnesium acetate) and heating at 94° C. for 2 min30 s. Fragmented RNA was purified using a Nucleospin RNA XS spin column(Macharey Nagel).

10 ng of fragmented RNA in 3.5 μl was combined with 1 μl 120 firststrand primer. Samples were heated to 72° C. for 3 minutes and thenplaced on ice. To these samples were added 2 μl 5× first strand buffer(Clontech), 0.25 μl 100 mM DTT, 0.25 μl recombinant RNase inhibitor(Takara), 10 mM dNTP mix (Clontech), 1 μl 120 template switch oligo, and1 μl SMARTScribe RT (Clontech). Samples were incubated at 42° C. for 90minutes followed by 70° C. for 10 minutes.

First strand cDNA reactions were purified by addition of 15 μl water and25 μl Ampure XP beads (Beckman Coulter). Samples were well mixed andincubated at room temperature for 8 minutes. Samples were bound to amagnetic stand for 5 minutes, and the beads were washed twice with 200μl 80% ethanol and allowed to air dry for 5 minutes.

cDNA on the beads was eluted by addition of 50 μl PCR Mastermix (5 μl 10Advantage2 buffer, 5 μl GC Melt reagent, 1 μl 10 mM dNTPs, 1 μlAdvantage2 polymerase (All Clontech), 240 nM forward PCR primer, 240 nMreverse PCR primer, and 36.8 μl water). Samples were thermocycled for 12PCR cycles with the settings 95° C. 1 minute, 12× (95° C. 15 seconds,65° C. 30 seconds, 68° C. 1 minute). PCR products were purified with 50μl Ampure XP beads and eluted in 40 μl TE buffer. Samples were dilutedand run on an Agilent Bioanalyzer using the high sensitivity DNA assay.

B. Sequencing

The above sequencing library was diluted to 2 nM and combined with anequal amount of PhiX Control Library (Illumina). Samples were loadedonto an Illumina MiSeq instrument with a final loading concentration of8 pM and sequenced as a single 66 bp read.

C. Analysis Summary

All Analysis was performed on a linux workstation. Sequences weretrimmed of the first three nucleotides, and PhiX sequences werebioinformatically removed by mapping all sequences to the PhiX genomewith the Bowtie2 software package and retaining all unmapped reads.

Remaining sequencing reads were mapped to the mouse transcriptome (buildMM10) using the tophat2 software package. Gene expression values werecalculated using the Cufflinks software using the genome annotation as aguide.

Gene expression values were compared to a previously sequenced librarygenerated with the SMARTer Universal kit (Clontech) from ribosomallydepleted Mouse Brain Total RNA (Clontech).

Gene expression comparisons and plotting were done In R using theCummeRbund analysis package.

Gene body coverage and strand specificity were calculated usinggeneBody_coverage.py and infer_experiment.py scripts respectively fromthe RSeQC software collection.

III. miRNA Library Construction

1 μl of 5 μM synthetic miR-22 (AAGCUGCCAGUUGAAGAACUGUA) (RNA) (SEQ IDNO:07) was combined with 2 μl 5× First Strand Buffer (Clontech), 0.25 μl100 mM DTT, 0.25 μl Recombinant RNase inhibitor (Takara), 0.25 μlPoly(A) polymerase (Takara), 1 μl 10 mM ATP, 5.25 μl RNase free water.Samples were incubated at 37° C. for 10 minutes followed by 65° C. for20 minutes.

Reactions were diluted with 10 μl RNase free water. 3.5 μl dilutedpolyadenylated miRNA was combined with 1 μl 12 μM first strand primer.Samples were heated to 72° C. for 3 minutes and then placed on ice. Tothese samples were added 2 μl 5× first strand buffer (Clontech), 0.25 μl100 mM DTT, 0.25 μl recombinant RNase inhibitor (Takara), 10 mM dNTP mix(Clontech), 1 μl 120 template switch oligo, and 1 μl SMARTScribe RT(Clontech). Samples were incubated at 42° C. for 60 minutes followed by70° C. for 15 minutes.

First strand reactions were diluted with 40 μl TE buffer. 5 μl dilutedcDNA was combined with 45 μl PCR Mastermix (5 μl 10 Advantage2 buffer, 1μl 10 mM dNTPs, 1 μl Advantage2 polymerase (All Clontech), 240 nMforward PCR primer, 240 nM reverse PCR primer (and 36 μl water). Sampleswere thermocycled for 20 PCR cycles with the settings 95° C. 1 minute,20× (95° C. 15 seconds, 65° C. 30 seconds). 5 μl PCR products wereresolved on a 1% agarose gel.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1-45. (canceled)
 46. A method comprising: (a) providing a precursor ribonucleic acid (RNA); (b) adding a plurality of non-templated nucleotides to an end of the precursor RNA to produce a template RNA; (c) combining: the template RNA; a template switch oligonucleotide (TSO) comprising a first nucleotide sequence domain; a polymerase having terminal transferase activity; a first strand primer comprising a first domain that hybridizes to the non-templated nucleotides in the template RNA and a second domain having a nucleotide sequence that is different from the first nucleotide sequence domain present in the TSO; and dNTPs; in a reaction mixture under conditions sufficient to produce a product nucleic acid comprising the template RNA and the TSO hybridized to adjacent regions of a single product nucleic acid comprising the first strand primer and a region polymerized from the dNTPs by the polymerase; and (d) amplifying the product nucleic acid with a forward primer and a reverse primer, wherein each of the forward primer and the reverse primer comprises a sequencing platform adapter construct comprising at least a portion of a capture sequence that is utilized by a sequencing platform, wherein the capture sequence specifically hybridizes to a surface-attached sequencing platform oligonucleotide on the sequencing platform, and wherein the forward and reverse primers are different and hybridize to different sequences of the product nucleic acid.
 47. The method according to claim 46, further comprising sequencing the amplified product nucleic acid by the sequencing platform that comprises the surface-attached sequencing platform oligonucleotide that captures the at least portion of the capture sequence of the sequencing platform adapter construct.
 48. The method according to claim 46, wherein the non-templated nucleotides are added to the 3′ end of the precursor RNA.
 49. The method according to claim 46, wherein the non-templated nucleotides are added in an enzymatic reaction.
 50. The method according to claim 46, wherein the non-templated nucleotides comprise a polyadenylation (poly A) sequence.
 51. The method according to claim 46, wherein the precursor RNA is a non-polyadenylated RNA.
 52. The method according to claim 51, wherein the non-polyadenylated RNA is selected from the group consisting of a microRNA, a siRNA, and a small RNA.
 53. The method according to claim 46, wherein the polymerase is a reverse transcriptase.
 54. The method according to claim 46, wherein the template switch oligonucleotide, the first strand primer, or both the template switch oligonucleotide and the first strand primer comprise a sequencing platform adapter construct that is utilized by a sequencing platform.
 55. The method according to claim 54, wherein the sequencing platform adapter construct comprises a nucleic acid domain selected from the group consisting of: a domain that specifically hybridizes to the surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, and combinations thereof.
 56. A kit, the kit comprising: a) a template switch oligonucleotide (TSO) comprising a 3′ hybridization domain; b) a first polymerase; c) a first strand primer comprising a first domain that hybridizes to a template RNA; d) dNTPs; and e) a second polymerase.
 57. The kit according to claim 56, wherein the first polymerase further comprises terminal transferase activity and template switching activity
 58. The kit according to claim 56, wherein the first polymerase is a reverse transcriptase.
 59. The kit according to claim 58, wherein the reverse transcriptase is a MMLV reverse transcriptase.
 60. The kit according to claim 56, wherein the second polymerase adds dNTPs to the 3′ end of a precursor RNA to produce the template RNA. 